作者要感谢劳拉·迪亚斯，凯文·菲利普斯，薇拉薛，王C.李的宝贵意见和技术支持，在发展这种方法。

1。动物的制备和冷处理<ol><li>找到并检查了4℃冷室中，已被批准用于容纳实验室小鼠。 </li><li>预冷动物的笼子，在寒冷的房间里过夜。网箱组装饲料和床上用品，但没有一瓶水。 </li><li>在的实验一天早上，地方小鼠一个由一个以30分钟的间隔到各预冷的笼子。每个单笼的鼠标应该留在寒冷的房间了近4个小时，然后才运到的成像实验室。确保小鼠禁食，但获得水。 </li><li>后4小时冷处理运输动物在同一时间，每30分钟的成像实验室。这可以通过以下来实现填充一个塑料容器中，用冰和冰内盒的顶部放置一个预冷冻的住房笼。松散地放置在封面上的泡沫塑料盒。 </li></ol> 2。设置Micro-PET/CT成像流程<p class=在该协议中micro-PET/CT成像的的"jove_content"&gt;实现与西门子Inveon专用PET（DPET）系统和的Inveon多模（MM）系统（CT / SPECT）在停靠模式。动物被置于从MM的入口，第一与解剖参考的CT扫描，然后滑动到中心的DPET静态F18 PET收集。为了使主机进行自动连续任务编程的的Inveon收购工作场所（IAW）软件的实际成像会议之前，下面的"工作流程"。 <ol><li> CT采集 :对于一个全身CT扫描，设置在500微安的电流，电压在80KV，曝光时间为200毫秒，240步，240°旋转。对于X射线探测器，在"低系统放大倍率为78毫米的轴向成像领域和一张单人床模式选择分辨率。选择"实时重建"使用"通用锥束重建"的方法，使主机PC会谈的专用的实时重建计算机（眼镜蛇）启动任务。 </li><li> PET发射采集 :设置为600秒（10分钟）在"获取时间"选项中的"固定扫描时间"。选择F-18"研究同位素"，并使用350-650千电子伏的能量水平"。 </li><li> PET排放直方图 :将"动态帧"，"黑"过程数据作为一帧的整个持续时间实现静态扫描。选择"3D"作为直方图类型，选择"不分散修正"。 </li><li> PET图像重建 :使用二维有序的子集期望最大化（OSEM2D）的重建算法。 </li></ol> 3。注射FDG <ol><li>订购的18 F-FDG（10 mCi）的临床包从当地供应商，它的到来〜30分钟之前预定的第一次注射的成像实验室。按照该机构的安全程序，以接收和调查的程序包，其中包含放射性本草的ls（RAM）。 </li><li>提供的L-块表顶盾，分装FDG，并用无菌生理盐水稀释的保护。 FDG的稀释活度浓度应可在200-300μCi/100UL，每次注射。绘制的葡萄糖溶液1毫升注射器26G 1/2寸毫针，并测量放射性的剂量校准的注射器。 </li><li>注入动物，只是输送FDG溶液与100μl通过腹腔内（IP）的路由从冷​​室（见步骤1.4）。记录的喷射时间。注射器的再次测量残余物的放射性剂量校准器。 </li><li>将动物的冷笼子内保持着冰保丽龙冷却器。孵育1小时FDG摄取动物在寒冷的（〜4°C）。 </li><li>计算各小鼠注入FDG活性由下式: 注射的活性（微居里）=活动注射器注射前- 注射器注射后活动</li></ol> 4。 Micro-PET/CT成像<ol><li> micro-PET/CT成像开始1小时后的葡萄糖注射液或5小时后的冷处理。将动物麻醉诱导室与3％异氟醚氧。 </li><li>麻醉诱导后，动物被移动到一个微型CT粒料（动物床）与它的头靠在一个锥形内面罩，不断提供异氟醚（2％），在2升/分钟的流速。电热垫（BioVet，M2M IMAGING CORP。）下的动物，以帮助维持体温。 </li><li>将动物的MM扫描仪的入口，启动"激光"从工具栏上的图标，并使用触摸板控制来移动床，让胸部的动物是在十字架上的水平和垂直的激光线。在"的激光对齐"窗口中，选择"先扫描类型"CT扫描，和"工作流程中的收购包括PET"的OPTION。 </li><li>打开"童军视图"窗口和取得的球探视图X射线摄片。使用IAW动物床的位置调整，使得它的中心的CT视图字段位于鼠标的中心体（肝脏）。如有必要，重复此步骤。 </li><li>启动在步骤2中建立的"工作流"。当选项出现，选择一个合适的3D PET-CT变换矩阵文件中使用的CT重建，并选择最近创建的文件没有衰减校正的PET重建正常化。 ，IAW将开始CT和PET扫描顺序编程。 </li><li>在整个工作流程完成，这可能需要20-25分钟，简单的质量评价所获得的CT和PET图像与ASIPro微型PET分析软件的虚拟机，安装了IAW。存档的摄像数据的数据存储装置，或通过网络将数据传输到后成像分析工作站（见步骤5），以供进一步分析。 </li><li> Relea从成像系统本身的动物，并将其放置到一个干净的壳体保持架，其在室温下的恢复正常的食物和水供应。该系统现在已经准备好为在队列中的下一个动物。请注意照顾和处理后成像的动物应遵守学院的规定"处理实验动物注射RAM"。另外还要注意所使用的针头/注射器，吸水垫，手套，和所有床上用品和排泄物应被视为放射性废物，并根据学院的有关RAM废弃物处理法规进行处理。 </li></ol> 5。后成像分析<ol><li>开放Inveon研究工作场所（IRW）软件（西门子）和手动导入CT和PET数据集。合作寄存器的CT和PET图像中的"注册"窗口"分析"功能使用的工具，并在"审阅"窗口中确保一个完美的CT和PET图像在三维空间之间的一致性。 </li><li>从"感兴趣区域（ROI）量化"的窗口，提供的参考共同注册的CT图像，确定在肩胛间区的脖子在成年小鼠中，最主要的冷诱导BAT BAT，绘制感兴趣体积（VOI）的英美烟草公司在PET数据集。 "量化类型"选择"体素灰度"和贝克/毫升的VOI内录得的平均放射性。校准系数转换成计数/秒贝克/毫升先前已建立了一个幽灵与已知的放射性扫描。 </li><li> FDG摄取BAT被量化为每克组织百分注射剂量（％ID / g）的衰减校正。的注射剂量是3.5的步骤的结果，然而，转换贝克（Bq）的单元（1微居里= 37,000 Bq）为，我们假设一个1毫升的组织等于1克。 </li></ol>

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没有利益冲突的声明。

在这项研究中的一个micro-PET/CT-based成像方法已发展为在成年小鼠中检测BAT活动只需要一个冷处理和注射市售18 F-FDG。整个的过程可以在一天内完成后开始，每30分钟，直到所有的动物处理和成像处理和成像顺序。共10只小鼠（或2组，每组5只小鼠），在实验条件下，可以在同一天进行测试，用一个单一的成像系统。如果2个或更多的动物可以在一个特别设计的动物床同时扫描，因为它先前已报道17，不限于这种类型的吞吐量可以解除。为了完成这项研究，我们依靠的组合micro-PET/CT的利用提供详细的解剖信息的CT成像系统的使用。然而，一个独立的微PET也能完成任务时，57 CO传输扫描被添加到之前的工作流程F18排放数据采集。可以使用的57有限公司发送数据进行衰减校正PET图像重建过程中，也可以重建解剖定位。 此协议的一个关键步骤是优化的治疗感冒的持续时间（ 例如在这项研究中，5小时）。一个较短的时间或消除冷暴露可能会产生一个活动的背景和方法可以不受任何下调的BAT（地面效应）。与此相反，细长的暴露于寒冷的环境（如过夜，或24小时），可最大限度地提高了归纳和方法可以掩盖上调BAT（天花板效应）的差异趋于饱和。在本协议中所述的优化条件下，已验证通过普萘洛尔抑制和β3受体激动剂BRL37344刺激测试（ 图2 </strONG&gt;），这表明，这种方法是相当敏感的，一致的，在检测小鼠BAT活动改变的。这种方法的应用将包括各种利用小鼠模型的基础研究，以便更好地了解英美烟草公司的分化和调控，以及临床前研究，旨在发现安全BAT-刺激性的药物，可能有利于治疗肥胖和糖尿病的。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> Micro-PET/CT成像系统</td><td>西门子医疗解决方案（美国）公司</td><td> Inveon专用PET系统和的Inveon多模式CT / SPECT系统（停靠） </td><td></td></tr><tr><td>普萘洛尔</td><td>西格玛</td><td> P0884 </td><td></td></tr><tr><td> BRL 37344 </td><td>西格玛</td><td> B169 </td><td></td></tr><tr><td> 18 F-FDG </td><td> Cyclotope公司</td><td></td><td></td></tr><tr><td> C57BL/6J雄性小鼠</td><td>杰克逊实验室</td><td> 000664 </td><td> 3-4个月</td></tr></tbody></table>

functional imaging of brown fat in mice with 18f-fdg micro-pet/ct

其离散的位置，由于丰富的血管和高密度的线粒体和棕红色，褐色脂肪组织（BAT）与白色脂肪组织（WAT）。在新生哺乳动物的能量消耗和非颤抖性产热以及成人1 BAT发挥了重要作用。 BAT-介导的产热是由交感神经系统高度管制的，主要是通过β肾上腺素能受体2，3。最近的研究显示，英美烟草公司活动呈负相关，与成人的身体质量指数（BMI）和其他糖尿病参数4-6。因此，英美烟草公司提出专注于调制的能量平衡6-8 anti-obesity/anti-diabetes治疗的潜在目标。几个冷的挑战正电子发射断层扫描（PET）方法，建立了用于检测人BAT 9-13，基本上是没有什么标准协议的成像的量化ication的小动物模型，如小鼠BAT。在这里，我们描述了一个强大的PET / CT成像方法在小鼠BAT功能评估。简单地说，成年小鼠冷处理在空腹状态下，持续时间4小时之前，他们收到了一剂18 F-脱氧葡萄糖（FDG）。将小鼠保持在寒冷的一个额外小时后FDG注射，然后一个小的动物专用micro-PET/CT系统与扫描。所收购的PET图像共同登记的的CT图像解剖引用，并分析了FDG摄取在肩胛间BAT区提出BAT活动。这种标准化的冷处理和成像协议已经验证通过测试BAT活动，例如，在药物干预的抑制的BAT活化治疗β-受体阻滞剂心得安，14，15，或增强BAT激活β3受体激动剂BRL37344 16。法德cribed在这里可以应用到屏幕的药物/化合物，调节BAT的活动，或确定基因/ BAT各种临床前研究和基础研究的发展和法规中所涉及的途径。

小鼠BAT micro-PET/CT成像的一个例子示出在图1中。虽然CT成像提供解剖信息，PET成像编码的18 F-FDG摄取的分布和数量在整个身体。这些成像数据可以被分开（图1A和图1B），稠合的（1C），如最大强度投影（MIP，1D）与3D特征或演示。随着三维成像工具，感兴趣的体积（VOI）的帮助下，肩胛间BAT区域（ 图1中箭头所示），在这里上绘制的PET图像和总信号内的VOI％ID可以被转换成/ g时，表示的百分比每克组织的注射剂量（％ID）。在小鼠证明，FDG摄取的BAT为19％ID /克。为了验证这个寒冷的感应和成像协议是敏感，可以探测改变BAT的活动，在这两种情况下，上调或下调，我们使用β肾上腺素能受体拮抗剂普萘洛尔抑制的BAT活化15，和β3激动剂BRL37344 BAT感应16，分别以加强。冷处理过程中应用这些药物干预的，准确的说，在注射FDG前30分钟。 PET / CT成像（ 图2A）和（ 图2B）的分析表明，普萘洛尔治疗的显着降低，而在BAT FDG摄取BRL37344显着升高的吸收，与车辆用控制相比。 <img src="/files/ftp_upload/4060/4060fig1.jpg" alt="图1"/> 图1。5小时冷处理后的小鼠BAT Micro-PET/CT成像。 （A）A冠部CT图像。 （B）A冠部注册的PET图像与CT在"A"。 （C）稠合PET / CT图像导致从重叠的"A"和"B"。 （D）最大强度投影法（MIP）介绍的PET / CT融合图像。黄色箭头:肩胛间的棕色脂肪组织相对应的区域。 <img src="/files/ftp_upload/4060/4060fig2.jpg" alt="图2"/> 图2。Micro-PET/CT示范肾上腺素受体 ​​药物的BAT活性的变化。 （A）18 F-FDG PET / CT成像不同药物治疗的小鼠。一）PBS（对照）。 B）普萘洛尔（β受体拮抗剂，5mg/kg体重，IP）。注意BAT面积显着减少的FDG摄取。 C）BRL37344（β3受体激动剂，5mg/kg体重，IP）。请注意FDG积聚在英美烟草公司的一个显着增加。黄色箭头:肩胛间的棕色脂肪组织相对应的区域。 （B）定量分析的FDG摄取BAT。值的％ID /克（每克组织百分注射剂量）（平均值±SD）。每组10的PBS组，每组5为普萘洛尔和BRL37344组。 *表示p &lt;0.05; **P &lt;0.005，与PBS对照组相比。

Watch this Scientific Journal Video about Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT at JoVE.com

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

功能成像的小鼠棕色脂肪组织（BAT）的方法进行了说明，冷刺激BAT摄取18F-脱氧葡萄糖（FDG）的非侵入性评估与标准化的micro-PET/CT协议。这种方法的鲁棒性和敏感的检测BAT活动在小鼠模型中的差异。

功能成像在小鼠体内的棕色脂肪与FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

functional-imaging-of-brown-fat-in-mice-with-18f-fdg-micro-petct

Nanyang Technological University

Research

JoVE Journal

Biology

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

19.0K 次观看.  The Methodist Hospital Research Institute, Houston. 功能成像的小鼠棕色脂肪组织（BAT）的方法进行了说明，冷刺激BAT摄取18F-脱氧葡萄糖（FDG）的非侵入性评估与标准化的micro-PET/CT协议。这种方法的鲁棒性和敏感的检测BAT活动在小鼠模型中的差异。

视频: Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

光/暗转换测试小鼠

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

科研

生物学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, simple perceptual tasks are combined with appetitive and aversive reinforcement.  However, the use of reinforcers such as juice and airpuffs can create challenges for fMRI.  Reinforcer delivery can cause head movement, which creates artifacts in the fMRI signal.  Reinforcement can also lead to changes in heart rate and respiration that are mediated by autonomic pathways.  Changes in heart rate and respiration can directly affect the fMRI (BOLD) signal in the brain and can be confounded with signal changes that are due to neural activity.  In this presentation, we demonstrate methods for administering reinforcers in a controlled manner, for stabilizing the head, and for measuring pulse and respiration.

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

加固，Eyetracking，生理监测的功能成像

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

对小鼠高架十字迷宫

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

传统美国宇航局后肢卸小鼠模型的一个交替的方法

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

映射后的Theta突发刺激对人类听觉皮层功能成像

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

基于光二极管的光学成像，用于记录非转基因无脊椎动物的单神经元分辨率的网络动态

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...